Process for the Production of Estetrol

ABSTRACT

The present invention relates to a process for the preparation of a compound of formula (I), hydrates or solvates thereof.

FIELD OF THE INVENTION

The present invention relates to a new process for the synthesis ofEstetrol.

BACKGROUND OF THE INVENTION

Estrogenic substances are commonly used in methods of HormoneReplacement Therapy (HRT) and methods of female contraception. Estetrolis a biogenic estrogen that is endogenously produced by the fetal liverduring human pregnancy. Recently, estetrol has been found effective asan estrogenic substance for use in HRT. Other important applications ofestetrol are in the fields of contraception, therapy of auto-immunediseases, prevention and therapy of breast and colon tumors, enhancementof libido, skin care, and wound healing.

The synthesis of estetrol and derivatives thereof is known in the art.J. FISHMAN and H. GUZIK (J. Org. Chem, Vol 33, No 8, 3133-3135, 1968)describe a route to estra-1,3,5(10)-triene-3,15α,16α,17β-tetrol(estetrol) involving cis hydroxylation of the double bond of anα-β-unsaturated dioxolane derivative of formula A, wherein Ac is acetyl.

Osmium tetraoxyde was used for the cis hydroxylation of compound (A) andgave the 17,17-ethylenedioxyestra-1,3,5(10)-triene-3,15α,16α-triol3-acetate as the major product. However attempts to remove the dioxolanegroup failed completely.

The carbonyl group at C₁₇ of the3-hydroxyestetra-1,3,5(10),15-tetraen-17-one was reduced with LiAlH₄ toestra-1,3,5(10),15-tetraene-3,17-diol that was isolated as the diacetate(compound B). Compound B was subjected to cis-hydroxylation of thedouble bond of D ring by using Osmium tetraoxyde which resulted into theformation of estra-1,3,5(10)-triene-3,15α,16α,17α-tetraol-3,17-diacetate(compound C) as the major product associated withestra-1,3,5(10)-triene-3,15β,16β,17β-tetrol-3,17 diacetate. Thesecompounds were isolated by thin layer chromatography. Compound C underheating with K₂ CO₃ in methanol produces estetrol (compound D) (Scheme1). The overall yield of this three step process was, starting fromestrone 3-hydroxyestetra-1,3,5(10),15-tetraen-17-one, only about 7%.

Verhaar M. T; et al (WO 2004/041839) describes a process for thepreparation of estetrol by cis hydroxylation of17-acetyloxy-3-benzyloxy-estra-1,3,5(10),15-tetraene using osmiumtetraoxyde and trimethyl-amine N-oxide in THF at 50° C. The resulting15,16-dihydroxylated crude derivative was obtained in 84% yield butseveral crystallizations were needed in order to purify thisintermediate. Finally the yield after these purifications was about 43%.

Bull, James R; et al in Journal of the Chemical Society, PerkinTransactions 1: Organic and Bio-Organic Chemistry (1972-1999), (2),241-51; 1990 described cis hydroxylation using osmium tetraoxyde on a14,17-ethano derivative of formula (E) wherein Pa is a methyl group andPb is an acetyl group. A mixture was obtained consisting of about 56% ofthe α,α-dihydroxy and 27% of the β,β-dihydroxy derivative.

Beside the poor selectivity for osmium-catalyzed dihydroxylation ofthese 17β-acetyloxy derivatives, exhaustive purifications are needed.

There remain a need for an improved synthesis of estra-1,3,5(10),15α,16α,17β-tetrol (estetrol).

It is therefore an object of the present invention to provide a processfor the preparation of estra-1,3,5(10)-triene 15α,16α,17β-tetrol whichovercome at least one the disadvantages of the prior art.

SUMMARY OF THE INVENTION

The present inventors have now found that this object can be obtained byusing a process as defined in the appended claims.

According to the present invention, a process for the preparation of acompound of formula (I) (estra-1,3,5(10)-triene-3,15α,16α,17β-tetrol) isprovided:

said process comprises the steps of:

a) reacting a compound of formula (II), with an acylating or asilylating agent to produce a compound of formula (III),

wherein P¹ is a protecting group selected from R¹CO—, or R²Si(R³)(R⁴)—,P² is a protecting group selected from (R⁶R⁵R⁷)C—CO—, or(R²)Si(R³)(R⁴)—, wherein R¹ is a group selected from C₁₋₆alkyl orC₃₋₆cycloalkyl, each group being optionally substituted by one or moresubstituents independently selected from fluoro or C₁₋₄alkyl; R², R³ andR⁴ are each independently a group selected from C₁₋₆alkyl or phenyl,each group being optionally substituted by one or more substituentsindependently selected from fluoro or C₁₋₄alkyl; R⁵ is a group selectedfrom C₁₋₆alkyl or phenyl, each group being optionally substituted by oneor more substituents independently selected from fluoro or C₁₋₄alkyl; R⁶and R⁷ are each independently hydrogen or a group selected fromC₁₋₆alkyl or phenyl, each group being optionally substituted by one ormore substituents independently selected from fluoro or C₁₋₄alkyl;

b) reacting the compound of formula (III) in the presence of at leastone oxidizing agent selected from permanganate salt, osmium oxide,hydrogen peroxide, or iodine and silver acetate to produce compound offormula (IV); and

c) deprotecting the compound of formula (IV) to produce compound offormula (I).

The invention provides an improved process for producing a compound offormula (I) in significantly higher yield and for at lower cost thanpossible by the previous known syntheses. In particular, the presentprocess allows the preparation ofestra-1,3,5(10)-triene-3,15α,16α,17β-tetrol as the major product withlittle or no estra-1,3,5(10)-triene-3,15β,16β,17β-tetrol isomer.

According to a second aspect, the present invention also encompassesestetrol directly obtained by the process according to the presentinvention, for use in a method selected from a method of hormonereplacement therapy, a method of treating vaginal dryness, a method ofcontraception, a method of enhancing libido, of method of treating skin,a method of promoting wound healing, and a method of treating orpreventing a disorder selected from the group consisting of autoimmunediseases, breast tumors and colorectal tumors.

The above and other characteristics, features and advantages of thepresent invention will become apparent from the following detaileddescription, which illustrate, by way of example, the principles of theinvention.

DETAILED DESCRIPTION OF THE INVENTION

It is also to be understood that the terminology used herein is notintended to be limiting, since the scope of the present invention willbe limited only by the appended claims.

As used herein, the singular forms “a”, “an”, and “the” include bothsingular and plural referents unless the context clearly dictatesotherwise.

The terms “comprising”, “comprises” and “comprised of” as used hereinare synonymous with “including”, “includes” or “containing”, “contains”,and are inclusive or open-ended and do not exclude additional,non-recited members, elements or method steps. It will be appreciatedthat the terms “comprising”, “comprises” and “comprised of” as usedherein comprise the terms “consisting of”, “consists” and “consists of”.

The recitation of numerical ranges by endpoints includes all numbers andfractions subsumed within the respective ranges, as well as the recitedendpoints.

All references cited in the present specification are herebyincorporated by reference in their entirety. In particular, theteachings of all references herein specifically referred to areincorporated by reference.

Unless otherwise defined, all terms used in disclosing the invention,including technical and scientific terms, have the meaning as commonlyunderstood by one of ordinary skill in the art to which this inventionbelongs. By means of further guidance, term definitions are included tobetter appreciate the teaching of the present invention.

In the following passages, different aspects of the invention aredefined in more detail. Each aspect so defined may be combined with anyother aspect or aspects unless clearly indicated to the contrary. Inparticular, any feature indicated as being preferred or advantageous maybe combined with any other feature or features indicated as beingpreferred or advantageous.

Reference throughout this specification to “one embodiment” or “anembodiment” means that a particular feature, structure or characteristicdescribed in connection with the embodiment is included in at least oneembodiment of the present invention. Thus, appearances of the phrases“in one embodiment” or “in an embodiment” in various places throughoutthis specification are not necessarily all referring to the sameembodiment, but may. Furthermore, the particular features, structures orcharacteristics may be combined in any suitable manner, as would beapparent to a person skilled in the art from this disclosure, in one ormore embodiments. Furthermore, while some embodiments described hereininclude some but not other features included in other embodiments,combinations of features of different embodiments are meant to be withinthe scope of the invention, and form different embodiments, as would beunderstood by those in the art. For example, in the appended claims, anyof the claimed embodiments can be used in any combination.

The term “alkyl” by itself or as part of another substituent, refers toa straight or branched saturated hydrocarbon group joined by singlecarbon-carbon bonds having 1 to 6 carbon atoms, for example 1 to 5carbon atoms, for example 1 to 4 carbon atoms, preferably 1 to 3 carbonatoms. When a subscript is used herein following a carbon atom, thesubscript refers to the number of carbon atoms that the named group maycontain. Thus, for example, C₁₋₆alkyl means an alkyl of one to sixcarbon atoms. Examples of alkyl groups are methyl, ethyl, propyl,isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, 2-methylbutyl, pentyliso-amyl and its isomers, hexyl and its isomers.

The term “C₃₋₆cycloalkyl”, as a group or part of a group, refers to asaturated or partially saturated cyclic alkyl radical containing fromabout 3 to about 6 carbon atoms. Examples of monocyclic C₃₋₆cycloalkylradicals include cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl.

The term “C₂₋₆alkenyl” by itself or as part of another substituent,refers to an unsaturated hydrocarbyl group, which may be linear, orbranched, comprising one or more carbon-carbon double bonds. Examples ofC₂₋₆alkenyl groups are ethenyl, 2-propenyl, 2-butenyl, 3-butenyl,2-pentenyl and its isomers, 2-hexenyl and its isomers, 2,4-pentadienyland the like.

The term “C₆₋₁₀aryl”, by itself or as part of another substituent, as agroup or part of a group, refers to a polyunsaturated, aromatichydrocarbyl group having a single ring (i.e. phenyl) or multiple ringsfused together (e.g. naphthalene), or linked covalently, typicallycontaining 6 to 10 atoms; wherein at least one ring is aromatic.Non-limiting examples of C₆₋₁₀aryl include phenyl (C₆aryl), naphthyl,indanyl, or 1,2,3,4-tetrahydro-naphthyl.

The term “C₁₋₆alkylcarbonyl”, as a group or part of a group, representsa group of Formula —CO—R^(a), wherein R^(a) is C₁₋₆alkyl as definedherein.

The term “C₃₋₆cycloalkylcarbonyl”, as a group or part of a group,represents a group of Formula —CO—R^(c), wherein R^(a) is C₃₋₆cycloalkylas defined herein.

The term “C₂₋₆alkenylC₁₋₆alkanoate” refers to a compound having theFormula R^(b)—O—CO—R^(a) wherein R^(a) is C₁₋₆alkyl as defined hereinand R^(b) is C₂₋₆alkenyl as defined herein.

The term “C₂₋₆alkenylC₃₋₆cycloalkanoate” refers to a compound having theFormula R^(b)—O—CO—R^(c) wherein R^(c) is C₃₋₆cycloalkyl as definedherein and R^(b) is C₂₋₆alkenyl as defined herein.

The term “C₁₋₆alkylenecarbonate” refers to a compound having the FormulaR^(b)—O—CO—O—R^(a) wherein R^(a) is C₁₋₆alkyl as defined herein andR^(b) is C₂₋₆alkenyl as defined herein.

The term “heteroaryl”, by itself or as part of another substituent,refers to an aromatic monocyclic or polycyclic heterocycle havingpreferably 5 to 7 ring atoms and more preferably 5 to 6 ring atoms,which contains one or more heteroatom ring members selected fromnitrogen, oxygen or sulfur. Non-limiting examples of a heteroarylinclude: pyridinyl, pyrrolyl, furanyl, thiophenyl, pyrazolyl,imidazolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, triazolyl,oxadiazolyl, thiadiazolyl, tetrazolyl, oxatriazolyl, thiatriazolyl,pyrimidyl, pyrazinyl, pyridazinyl, oxazinyl, dioxinyl, thiazinyl,triazinyl. Preferably heteroaryl is selected from the group comprisingpyridinyl, pyrrolyl, furanyl, thiophenyl, imidazolyl, pyrazolyl,oxazolyl, thiazolyl, and pyrazinyl. More preferably heteroaryl ispyridinyl.

The present invention relates to a process for preparing compound offormula (I); wherein said process comprises the steps of

a) reacting a compound of formula (II), with an acylating or asilylating agent to produce a compound of formula (III),

wherein

P¹ is a protecting group selected from R¹CO—, or R²Si(R³)(R⁴)—,

P² is a protecting group selected from (R⁶R⁵R⁷)C—CO—, or(R²)Si(R³)(R⁴)—,

R¹ is a group selected from C₁₋₆alkyl or C₃₋₆cycloalkyl, each groupbeing optionally substituted by 1, 2 or 3 substituents independentlyselected from fluoro or C₁₋₄alkyl; preferably R¹ is selected from thegroup comprising methyl, ethyl, propyl, isopropyl, butyl, isobutyl,tert-butyl, cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl, eachgroup being optionally substituted by 1, 2 or 3 substituentsindependently selected from fluoro or C₁₋₄alkyl; more preferably R¹ ismethyl, ethyl, propyl, isopropyl, cyclopentyl, or cyclohexyl, yet morepreferably R¹ is methyl, or ethyl;

R², R³ and R⁴ are each independently a group selected from C₁₋₆alkyl orphenyl, each group being optionally substituted by one or moresubstituents independently selected from fluoro or C₁₋₄alkyl; preferablyR², R³ and R⁴ are each independently selected from the group comprisingmethyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, andphenyl, each group being optionally substituted with 1, 2 or 3substituents each independently selected from fluoro or C₁₋₄alkyl;preferably R², R³ and R⁴ are each independently selected from the groupcomprising methyl, ethyl, propyl, isopropyl, or tert-butyl, and phenyl,each group being optionally substituted with 1, 2 or 3 substituents eachindependently selected from fluoro or C₁₋₂alkyl;

R⁵ is a group selected from C₁₋₆alkyl or phenyl, each group beingoptionally substituted by one or more substituents independentlyselected from fluoro or C₁₋₄alkyl; preferably R⁵ is selected from thegroup comprising methyl, ethyl, propyl, isopropyl, butyl, isobutyl,tert-butyl, and phenyl, each group being optionally substituted with 1,2 or 3 substituents each independently selected from fluoro orC₁₋₄alkyl; preferably R⁵ is selected from the group comprising methyl,ethyl, propyl, isopropyl, or tert-butyl, and phenyl, each group beingoptionally substituted with 1, 2 or 3 substituents each independentlyselected from fluoro or C₁₋₂alkyl;

R⁶ and R⁷ are each independently hydrogen or a group selected fromC₁₋₆alkyl or phenyl, each group being optionally substituted by one ormore substituents independently selected from fluoro or C₁₋₄alkyl;preferably R⁶ and R⁷ are each independently hydrogen or are selectedfrom the group comprising methyl, ethyl, propyl, isopropyl, butyl,isobutyl, tert-butyl, and phenyl, each group being optionallysubstituted with 1, 2 or 3 substituents each independently selected fromfluoro or C₁₋₄alkyl; preferably R⁶ and R⁷ are each independentlyhydrogen or a group selected from methyl, ethyl, propyl, isopropyl, ortert-butyl, and phenyl, each group being optionally substituted with 1,2 or 3 substituents each independently selected from fluoro orC₁₋₂alkyl;

said process also comprises the steps of:

b) reacting the compound of formula (III) in the presence of at leastone oxidizing agent selected from permanganate salt, osmium oxide,hydrogen peroxide, or iodine and silver acetate to produce compound offormula (IV); preferably said oxidizing agent is potassium permanganate;and

c) deprotecting the compound of formula (IV) to produce compound offormula (I).

In an embodiment, P¹ is R²Si(R³)(R⁴)—. Preferably P¹ is selected fromthe group comprising tert-butyl-dimethyl-silyl, diphenyl-methyl-silyl,dimethyl-phenyl-silyl, trimethyl-silyl, triethyl-silyl andtriisopropyl-silyl, each group being optionally substituted by one ormore substituents independently selected from fluoro or C₁₋₄alkyl; morepreferably P¹ is tert-butyl-dimethyl-silyl.

According to the invention, step (a) comprises reacting a compound offormula (II), with an acylating or a silylating agent to produce acompound of formula (III),

In an embodiment, compound of formula (II) can be reacted with asilylating agent and P² is R²Si(R³)(R⁴)—. Preferably P² is selected fromthe group comprising tert-butyl-dimethyl-silyl, diphenyl-methyl-silyl,dimethyl-phenyl-silyl, trimethyl-silyl, triethyl-silyl andtriisopropyl-silyl, each group being optionally substituted by one ormore substituents independently selected from fluoro or C₁₋₄alkyl; morepreferably P² is tert-butyl-dimethyl-silyl.

In an embodiment, P¹ and P² are each independently R²Si(R³)(R⁴)—.

Non-limiting examples of suitable silylating agent can be selected fromthe group comprising C₁₋₆alkylsilylchloride, C₁₋₆alkylsilyltriflate,C₆arylsilylchloride, C₆arylsilyltriflate, C₁₋₆alkylC₆arylsilylchloride,and C₁₋₆alkylC₆arylsilyltriflate, each group being optionallysubstituted by one or more substituents independently selected fromfluoro or C₁₋₄alkyl.

For example, formation of protected compound of formula (III) can beperformed by reaction of compound of formula (II) with a silylatingagent such as tert-butyl dimethylsilylchloride,diphenylmethylsilylchloride, dimethylphenylsilylchloride,trimethylsilylchloride, triethylsilylchloride, ortriisopropylsilylchloride, or such as tert-butyl dimethylsilyltriflate,diphenylmethylsilyltriflate, dimethylphenylsilyltriflate,trimethylsilyltriflate, triethylsilyltriflate, ortriisopropylsilyltriflate. The reaction can be performed in the presenceof a suitable base such as imidazole, 2,6-lutidine, collidine,triethylamine, or 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU). The reactioncan be performed at room temperature or under reflux. The reaction canbe performed in the presence of a suitable solvent such asdimethylformamide, dichloromethane, or toluene, or a mixture thereof.

In an embodiment, compound of formula (II) can be reacted with asilylating agent and P² is (R⁶R⁵R⁷)C—CO—. Preferably P² is terbutyl-CO.

In an embodiment, P¹ and P² are each independently (R⁶R⁵R⁷)C—CO—.

Non-limiting examples of suitable acylating agent can be selected fromthe group comprising

preferably

wherein R⁵, R⁶, R⁷ have the same meaning as that defined in claim 1, R⁸is a group selected from C₁₋₆alkyl, or C₂₋₆alkenyl, each group beingoptionally substituted by one or more substituents independentlyselected from fluoro or C₁₋₄alkyl.

Preferably, the acylating agent can be selected from the groupcomprising pivaloyl chloride, pivaloyl anhydride and the like.

The acylation when performed with acylating agent such asC₂₋₆alkenyl-ter-butyrate, can be performed in the presence of an acid,such as in the presence of sulfuric acid, or in the presence of aC₆₋₁₀arylsulfonic acid, optionally substituted by one or more chlorosubstituents. Non-limiting examples of a suitable acid includepara-toluene sulfonic acid, and sulfuric acid.

The acylation when performed with

can be performed in the presence of an organic base, such as imidazole,triethylamine and the like.

Step (b) can comprise reacting the compound of formula (III) in thepresence of at least one oxidizing agent selected from permanganatesalt, osmium oxide, or hydrogen peroxide, or iodine and silver acetateor ruthenium salt to produce compound of formula (IV).

Preferably, step (b) comprises reacting the compound of formula (III) inthe presence of at least one oxidizing agent selected from permanganatesalt, osmium oxide, or hydrogen peroxide, or iodine and silver acetateto produce compound of formula (IV).

This reaction can be performed in the presence of a co-oxidant such astrimethylamine n-oxide, quinuclidine N-oxide, N-methylmorpholineN-oxide, potassium ferricyanide, tert-butylhydroperoxide, or a phasetransfer catalyst such as tetralkylammonium salts.

Preferably step (b) is performed in the presence of a permanganate salt,such as potassium permanganate. The reaction can be performed in thepresence of a suitable acid such as formic acid. The reaction can beperformed at low temperature such as temperature below 10° C.,preferably below 5° C., preferably around 0° C. The reaction can beperformed in the presence of a suitable solvent such as acetone.

According to the invention, step (c) comprises deprotecting the compoundof formula (IV) to produce compound of formula (I).

Suitable methods and conditions for deprotecting compound of formula(IV), will be clear to the skilled person and are generally described inthe standard handbooks of organic chemistry, such as Greene and Wuts,“Protective groups in organic synthesis”, 3^(rd) Edition, Wiley andSons, 1999, which is incorporated herein by reference in its entirety.

For example, when P¹ and P² are each independently R²Si(R³)(R⁴)—, thedeprotection can be performed in the presence of a suitable acid, suchas hydrochloric acid, acetic acid and the like, or by employingstoechiometric amount of a tetralkyl ammonium fluoride derivative in asolvent.

For example, when P¹ and P² are each independently (R⁶R⁵R⁷)C—CO—, thedeprotection can be performed in the presence of a suitable acid, baseor reducing agents. Preferably, the deprotection can be performed usinga suitable base such as potassium carbonate, for example in methanol.

The compound of formula (II) can be obtained according to method knownto the skilled man in the art.

In an embodiment, compound of formula (II) can be prepared by a processcomprising the steps of:

i) reacting a compound of formula (V), with an acylating or a silylatingagent to produce a compound of formula (VI),

wherein P³ is a protecting group selected from R⁹CO—, orR¹⁰Si(R¹¹)(R¹²)—,

R⁹ is a group selected from C₁₋₆alkyl or C₃₋₆cycloalkyl, each groupbeing optionally substituted by one or more substituents independentlyselected from fluoro or C₁₋₄alkyl; preferably R⁹ is a group selectedfrom C₁₋₆alkyl or C₃₋₆cycloalkyl, each group being optionallysubstituted by 1, 2 or 3 substituents independently selected from fluoroor C₁₋₄alkyl; preferably R⁹ is selected from the group comprisingmethyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl,cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl, each group beingoptionally substituted by 1, 2 or 3 substituents independently selectedfrom fluoro or C₁₋₄alkyl; more preferably R⁹ is methyl, ethyl, propyl,isopropyl, cyclopentyl, or cyclohexyl, yet more preferably R⁹ is methyl,or ethyl;

R¹⁰, R¹¹ and R¹² are each independently a group selected from C₁₋₆alkylor phenyl, each group being optionally substituted by one or moresubstituents independently selected from fluoro or C₁₋₄alkyl; preferablyR¹⁰, R¹¹ and R¹² are each independently a group selected from C₁₋₆alkylor C₆aryl, said C₁₋₆alkyl or C₆aryl, being optionally substituted with1, 2 or 3 substituents independently selected from fluoro or C₁₋₆alkyl;preferably R¹⁰, R¹¹, and R¹² are each independently selected from thegroup comprising methyl, ethyl, propyl, isopropyl, butyl, isobutyl,tert-butyl, and phenyl, each group being optionally substituted with 1,2 or 3 substituents each independently selected from fluoro orC₁₋₄alkyl; preferably R¹⁰, R¹¹ and R¹² are each independently selectedfrom the group comprising methyl, ethyl, propyl, isopropyl, ortert-butyl, and phenyl, each group being optionally substituted with 1,2 or 3 substituents each independently selected from fluoro orC₁₋₂alkyl,

ii) reacting the compound of formula (VI) in the presence of palladiumacetate or a derivative thereof, or iodine (V) species, to producecompound of formula (VII); and

iii) reacting the compound of formula (VII) with a reducing agent toproduce compound of formula (II).

Preferably, compound of formula (II) can be prepared by a processcomprising the steps of:

i) reacting a compound of formula (V), with an acylating or a silylatingagent to produce a compound of formula (VI),

wherein P³ is a protecting group selected from R⁹CO—, orR¹⁰Si(R¹¹)(R¹²)—,

R⁹ is a group selected from C₁₋₆alkyl or C₃₋₆cycloalkyl, each groupbeing optionally substituted by one or more substituents independentlyselected from fluoro or C₁₋₄alkyl; preferably R⁹ is a group selectedfrom C₁₋₆alkyl or C₃₋₆cycloalkyl, each group being optionallysubstituted by 1, 2 or 3 substituents independently selected from fluoroor C₁₋₄alkyl; preferably R⁹ is selected from the group comprisingmethyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl,cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl, each group beingoptionally substituted by 1, 2 or 3 substituents independently selectedfrom fluoro or C₁₋₄alkyl; more preferably R⁹ is methyl, ethyl, propyl,isopropyl, cyclopentyl, or cyclohexyl, yet more preferably R⁹ is methyl,or ethyl;

R¹⁰, R¹¹ and R¹² are each independently a group selected from C₁₋₆alkylor phenyl, each group being optionally substituted by one or moresubstituents independently selected from fluoro or C₁₋₄alkyl; preferablyR¹⁰, R¹¹ and R¹² are each independently a group selected from C₁₋₆alkylor C₆aryl, said C₁₋₆alkyl or C₆aryl, being optionally substituted with1, 2 or 3 substituents independently selected from fluoro or C₁₋₆alkyl;preferably R¹⁰, R¹¹ and R¹² are each independently selected from thegroup comprising methyl, ethyl, propyl, isopropyl, butyl, isobutyl,tert-butyl, and phenyl, each group being optionally substituted with 1,2 or 3 substituents each independently selected from fluoro orC₁₋₄alkyl; preferably R¹⁰, R¹¹ and R¹² are each independently selectedfrom the group comprising methyl, ethyl, propyl, isopropyl, ortert-butyl, and phenyl, each group being optionally substituted with 1,2 or 3 substituents each independently selected from fluoro orC₁₋₂alkyl.

ii) reacting the compound of formula (VI) in the presence of palladiumacetate or a derivative thereof to produce compound of formula (VII);and

iii) reacting the compound of formula (VII) with a reducing agent toproduce compound of formula (II).

In an embodiment, P¹ is R¹CO—; preferably P¹ is a group selected fromC₁₋₄alkylcarbonyl or C₄₋₆cycloalkylcarbonyl, each group being optionallysubstituted by 1, 2 or 3 substituents independently selected from fluoroor C₁₋₄alkyl; more preferably P¹ is a group selected fromC₁₋₂alkylcarbony or C₅₋₆cycloalkylcarbonyl, each group being optionallysubstituted by 1, 2 or 3 substituents independently selected from fluoroor C₁₋₂alkyl; for example P¹ is selected from acetyl, ter-butyl-CO—, orcyclohexylcarbonyl, preferably P¹ is acetyl.

In an embodiment, P³ is R⁹CO—; preferably P³ is a group selected fromC₁₋₄alkylcarbonyl or C₄₋₆cycloalkylcarbonyl, each group being optionallysubstituted by 1, 2 or 3 substituents independently selected from fluoroor C₁₋₄alkyl; more preferably P³ is a group selected fromC₁₋₂alkylcarbony or C₅₋₆cycloalkylcarbonyl, each group being optionallysubstituted by 1, 2 or 3 substituents independently selected from fluoroor C₁₋₄alkyl; for example P³ is selected from acetyl, orcyclohexylcarbonyl, preferably P³ is acetyl.

In an embodiment, P¹ is R¹CO— and P³ is R⁹CO—. In an another embodiment,P¹ is R²Si(R³)(R⁴)—. Preferably P¹ is selected from the group comprisingtert-butyl-dimethyl-silyl, diphenyl-methyl-silyl, dimethyl-phenyl-silyl,trimethyl-silyl, triethyl-silyl and triisopropyl-silyl, each group beingoptionally substituted by one or more substituents independentlyselected from fluoro or C₁₋₄alkyl; more preferably P¹ istert-butyl-dimethyl-silyl.

In an embodiment, step (i) comprises the steps of (i1) protecting thehydroxyl of compound of formula (V) with a silylating agent to produce acompound of formula (Va), wherein P¹ has the same meaning as thatdefined herein above, preferably wherein P¹ is R²Si(R³)(R⁴)—; and

(i2) protecting the ketone of compound of formula (Va) in the presenceof an acylating agent to produce compound of formula (VI), preferablywherein P³ is R⁹CO—.

In an embodiment, P³ is R¹⁰Si(R¹¹)(R¹²)—; preferably P³ is selected fromthe group comprising tert-butyl-dimethyl-silyl, diphenyl-methyl-silyl,dimethyl-phenyl-silyl, trimethyl-silyl, triethyl-silyl andtriisopropyl-silyl, each group being optionally substituted by one ormore substituents independently selected from fluoro or C₁₋₄alkyl, morepreferably P³ is tert-butyl-dimethyl-silyl.

In an embodiment, P¹ is R²Si(R³)(R⁴)— and P³ is R¹⁰Si(R¹¹)(R¹²)—.

In another embodiment, P¹ is R²Si(R³)(R⁴)—; and P³ is R⁹CO—. PreferablyP¹ is selected from the group comprising tert-butyl-dimethyl-silyl,diphenyl-methyl-silyl, dimethyl-phenyl-silyl, trimethyl-silyl,triethyl-silyl or triisopropyl-silyl, each group being optionallysubstituted by one or more substituents independently selected fromfluoro or C₁₋₄alkyl; more preferably P¹ is tert-butyl-dimethyl-silyl;and preferably P³ is a group selected from C₁₋₆alkylcarbonyl orC₃₋₆cycloalkylcarbonyl, each group being optionally substituted by 1, 2or 3 substituents independently selected from fluoro or C₁₋₄alkyl;preferably P³ is a group selected from C₁₋₄alkylcarbonyl orC₅₋₆cycloalkylcarbonyl; each group being optionally substituted by 1, 2or 3 substituents independently selected from fluoro or C₁₋₂alkyl; morepreferably P³ is C₁₋₂alkylcarbony or C₅₋₆cycloalkylcarbonyl, for exampleP³ is acetyl or cyclohexylcarbonyl, preferably acetyl.

Suitable silylating agents and conditions are the same as describedherein above for step (a) of the process of the invention.

In an embodiment, wherein P¹ is R¹CO— and P³ is R⁹CO—, estrone can bereacted with an acylating agent. Preferably, said acylating agent isC₂₋₆alkenylC₁₋₆alkanoate or C₂₋₆alkenylC₃₋₆cycloalkanoate. Preferably,the acylating agent is selected from the group comprisingC₂₋₆alkenylpropanoate, C₂₋₆alkenylbutanoate, C₂₋₆alkenylpentanoate,C₂₋₆alkenylhexanoate, C₂₋₆alkenylcyclopropanoate,C₂₋₆alkenylcyclobutanoate, C₂₋₆alkenylcyclopentanoate, andC₂₋₆alkenylcyclohexanoate. More preferably, the acylating agent isselected from the group comprising isopropenyl acetate, isopropenylpropionate, isopropenyl butyrate, isopropenyl isobutyrate, vinylacetate, vinyl propionate, prop-2-enyl cyclohexanecarboxylate, ethenylcyclopentanecarboxylate, and vinyl cyclohexanoate. More preferably, theacylating agent is selected from the group comprising isopropenylacetate, isopropenyl propionate, isopropenyl butyrate, isopropenylisobutyrate, vinyl acetate, and vinyl propionate. The acylation can beperformed in the presence of an acid, such as in the presence ofsulfuric acid, or in the presence of an C₆₋₁₀arylsulfonic acid,optionally substituted by one or more chloro substituents. Non-limitingexamples of a suitable acid include para-toluene sulfonic acid, andsulfuric acid. For example, estrone of formula (V) can be was reactedwith isopropenyl acetate in the presence of sulfuric acid orpara-toluene sulfonic acid to give the estra-1,3,5 (10),16-tetraene-3,17-diol, 3,17-diacetate. The reaction can be performedunder reflux, optionally under inert atmosphere, such as nitrogenatmosphere. The product can be used as such in the next step or furtherpurified by known techniques in the art such as by chromatography, forexample on silica with a suitable eluant such as methylenechloride/hexane or ethyl acetate/hexane.

In an embodiment, wherein P¹ is R²Si(R³)(R⁴)— and P³ isR¹⁰Si(R¹¹)(R¹²)—, estrone of formula (V) can be reacted with asilylating agent. The silylating agent can be selected from the groupcomprising C₁₋₆alkylsilyl triflate, C₆arylsilyltriflate,C₁₋₆alkylC₆arylsilyltriflate, each group being optionally substituted byone or more substituents independently selected from fluoro orC₁₋₄alkyl. For example, formation of protected estrone silyl ether canbe performed by reaction of a silylating agent such as tert-butyldimethylsilyltriflate, diphenylmethylsilyltriflate,dimethylphenylsilyltriflate, trimethylsilyltriflate,triethylsilyltriflate, or triisopropylsilyltriflate. The reaction can beperformed in the presence of a suitable base such as imidazole,2,6-lutidine, collidine, triethylamine, or1,8-diazabicyclo[5.4.0]undec-7-ene (DBU). The reaction can be performedat room temperature or under reflux. The reaction can be performed inthe presence of a suitable solvent such as dichloromethane, toluene ordimethylformamide or a mixture thereof.

Step (ii) of the process for preparing compound of formula (II)comprises reacting the compound of formula (VI) in the presence ofpalladium acetate or a derivative thereof such as palladium chloride toproduce a compound of formula (VII).

In an embodiment, said palladium acetate can be present instoichiometric amounts, or sub-stoichiometric catalytic amounts. Forexample the reaction of step (ii) can be performed using stoichiometricamounts of palladium acetate, preferably in a suitable solvent suchbenzonitrile. This reaction can be performed at room temperature.

In another example, said step (ii) can be performed usingsub-stoichiometric catalytic amounts of palladium acetate in thepresence of a C₁₋₆alkylene carbonate such as allyl carbonate and in thepresence of an organotin compound as catalyst. Preferably, the organotincompound is tri-butyltin methoxide. Preferably the C₁₋₆alkylenecarbonate is allyl methyl carbonate. The reaction can be performed underreflux conditions, optionally under inert atmosphere such as nitrogen orargon atmosphere.

In another example, said step (ii) can be performed usingsub-stoichiometric catalytic amounts of palladium acetate under anoxygen atmosphere.

Alternatively, step (ii) of the process for preparing compound offormula (II) comprises reacting the compound of formula (VI) in thepresence of iodine (V) species.

Preferably, said iodine (V) species are selected from o-iodobenzoic acid(IBX also known as 1-hydroxy-1,2-benziodoxal-3(1H)-one-1-oxide) or IBXcomplexes, such as IBX•N-oxide complexes. Non-limiting examples ofsuitable IBX complexes include IBX-4-methoxypyridine-N-oxide complex(IBX•MPO complex), and complexes as described in Nicolaou et al. Angew.Chem. Int. Ed. 2002, 41, 996-1000 and Angew. Chem. Int. Ed. 2002, 41,993-995 hereby incorporated by reference in their entirety.

In another, more preferred embodiment, the iodine (V) species areselected from HIO₃ or/and its anhydride I₂O₅. These iodine (V) specieshave the advantage of being mild, safe and chemoselective reagentsavailable at reasonable cost for industrial applications.

Preferably, the oxidation with the iodine (V) species is carried out inthe presence of a ligand such as tetrahydrofuran (THF),dimethylsulfoxide (DMSO) or N-oxide derivatives such asN-methylmorpholine-N-oxide, 4-methoxypyridine-N-oxide,trimethylamine-N-oxide.

Preferably, the reaction is performed in the presence of a solvent, suchas DMSO. In an embodiment the reaction is kept at 45-65° C. Preferablythe reaction is performed at a temperature ranging from 45 to 65° C. inthe presence of DMSO.

The next step (iii) in the process comprises the reduction of thecompound of formula (IV) with a reducing agent to produce compound offormula (II). Preferably, said reducing agent is a metal hydridecompound. For example, the metal hydride compound can be selected fromthe group comprising LiAlH₄, NaBH₄, NaBH(OAc)₃, ZnBH₄, and NaBH₄/CeCl₃.preferably, said reducing agent is NaBH₄/CeCl₃. For example saidreduction can be performed in a suitable solvent or a mixture thereof,such as in tetrahydrofuran, or a mixture of methanol andtetrahydrofuran. The reaction can be performed at low temperatures suchas below 15° C., for example below 10° C.

In an embodiment, compound of formula (VII) is not isolated but directlyreduced to the alcohol using said reducing agent. In this embodiment,step (ii) and (iii) are performed in one pot. This one-pot/two-stepprocedure is the shortest chemical pathway described to obtain compoundof formula (II).

According to another embodiment, step (i) can be performed in two stepsand comprises the steps of (i1) protecting the hydroxyl of compound offormula (V) using a silylating agent to produce a compound of formula(Va), wherein P¹ is R²Si(R³)(R⁴)—; and

(i2) converting the ketone of compound of formula (Va) to its enol etherin the presence of an acylating agent to produce a compound of formula(VI).

In this embodiment, wherein P¹ independently R²Si(R³)(R⁴)—, and P³ isR⁹—CO—, estrone of formula (V) is reacted with a silylating agent toproduce compound of formula (Va). The silylating agent can be selectedfrom the group comprising C₁₋₆alkylsilyl chloride, C₆arylsilyl chloride,C₁₋₆alkylC₆arylsilyl chloride; each group being optionally substitutedby one or more substituents independently selected from fluoro orC₁₋₄alkyl. For example, formation of protected estrone silyl ether canbe performed by reaction of a silylating agent such as tert-butyldimethylsilylchloride, diphenylmethylsilylchloride,dimethylphenylsilylchloride, trimethylsilylchloride,triethylsilylchloride, or triisopropylsilylchloride. The reaction can beperformed in the presence of a base such as imidazole, 2,6-lutidine,collidine, triethylamine, or 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU).

The next step comprises, converting the ketone of compound of formula(Va) in the presence of an acylating agent to produce a compound offormula (VI) wherein P³ is acyl. Suitable acylating agents andconditions are as described herein above. The next step can comprisereacting the formula (VI) wherein P³ is acyl in the presence ofpalladium acetate or a derivative thereof to produce compound of formula(VII) wherein P¹ is R²Si(R³)(R⁴)—. This reaction can be performed asdescribed herein above. The next step in the process comprises thereduction of the compound of formula (VII) with a reducing agent toproduce compound of formula (II) wherein P¹ is R²Si(R³)(R⁴)—. Thisreaction can be performed as described herein above.

In another embodiment, compound of formula (II) can be prepared by aprocess comprising the steps of:

1) reacting a compound of formula (V) with a silylating or an acylatingagent to produce compound of formula (Va), wherein P¹ has the samemeaning as in claim 1;

2) halogenation or sulfinylation of the compound of formula (Va) toproduce a compound of formula (Vb);

wherein X is halo, or —O—SO—R²⁰, and R²⁰ is a group selected fromC₆₋₁₀aryl or heteroaryl, each group being optionally substituted by oneor more substituents independently selected from chloro or C₁₋₄alkyl;

3) dehalogenation or desulfinylation of the compound of formula (Vb) toproduce compound of formula (V); and

4) reacting the compound of formula (VII) with a reducing agent toproduce compound of formula (II).

According to step (1) of this embodiment, the hydroxyl of estrone offormula (V) is protected, to produce compound of formula (Va). In anembodiment, estrone of formula (V) can reacted with a silylating agent.Non-limiting examples of suitable silylating agents and conditions arethe same as described herein above for step (a) of the process of theinvention. For example, formation of protected estrone silyl ether canbe performed by reaction of a silylating agent such as tert-butyldimethylsilylchloride, diphenylmethylsilylchloride,dimethylphenylsilylchloride, trimethylsilylchloride,triethylsilylchloride, or triisopropylsilylchloride, or such astert-butyl dimethylsilyltriflate, diphenylmethylsilyltriflate,dimethylphenylsilyltriflate, trimethylsilyltriflate,triethylsilyltriflate, or triisopropylsilyltriflate. The reaction can beperformed in the presence of a suitable base such as imidazole,2,6-lutidine, collidine, triethylamine, or1,8-diazabicyclo[5.4.0]undec-7-ene (DBU). The reaction can be performedat room temperature or under reflux. The reaction can be performed inthe presence of a suitable solvent such as dichloromethane, toluene, ordimethylformamide or a mixture thereof.

In another embodiment, estrone of formula (V) can be reacted with anacylating agent. In an embodiment, said acylating agent can be selectedfrom the group comprising C₂₋₆alkenylC₁₋₆alkanoate,C₂₋₆alkenylC₃₋₆cycloalkanoate, acyl chloride, and anhydrides.Preferably, the acylating agent is selected from the group comprisingC₂₋₆alkenylpropanoate, C₂₋₆alkenylbutanoate, C₂₋₆alkenylpentanoate,C₂₋₆alkenylhexanoate, C₂₋₆alkenylcyclopropanoate,C₂₋₆alkenylcyclobutanoate, C₂₋₆alkenylcyclopentanoate, andC₂₋₆alkenylcyclohexanoate, acyl chloride and anhydrides. Morepreferably, the acylating agent is selected from the group comprisingisopropenyl acetate, isopropenyl propionate, isopropenyl butyrate,isopropenyl isobutyrate, vinyl acetate, vinyl propionate, prop-2-enylcyclohexanecarboxylate, ethenyl cyclopentanecarboxylate, vinylcyclohexanoate, acetyl chloride, propionylchloride, butyrylchloride,acetic anhydride and the like. More preferably, the acylating agent isselected from the group comprising isopropenyl acetate, isopropenylpropionate, isopropenyl butyrate, isopropenyl isobutyrate, vinylacetate, vinyl propionate, acetyl chloride, propionylchloride,butyrylchloride, acetic anhydride and the like. The acylation whenperformed with C₂₋₆alkenylC₁₋₆alkanoate orC₂₋₆alkenylC₃₋₆cycloalkanoate, can be performed in the presence of anacid, such as in the presence of sulfuric acid, or in the presence of aC₆₋₁₀arylsulfonic acid, optionally substituted by one or more chlorosubstituents. Non-limiting examples of a suitable acid includepara-toluene sulfonic acid, and sulfuric acid. The acylation whenperformed with an acyl chloride or an anhydride, can be performed in thepresence of an organic base, such as imidazole, triethylamine and thelike.

Step (2) of the process comprises halogenation or sulfinylation of thecompound of formula (Va) to produce a compound of formula (Vb); whereinX is halo, or —O—SO—R²⁰, and R²⁰ is a group selected from C₆₋₁₀aryl orheteroaryl, each group being optionally substituted by one or moresubstituents independently selected from chloro or C₁₋₄alkyl; preferablyR²⁰ is phenyl or pyridinyl.

In an embodiment, step (2) is a halogenation and the halogenation isperformed by reacting the compound of formula (Va) with a halogenatingreagent. Preferably, step 2) is a bromination, and X is bromo. In anembodiment, the brominating reagent can be selected from the groupcomprising copper(II) bromide, bromine, pyridine bromine perbromine andthe like.

In another embodiment, step (2) is a sulfinylation and the sulfinylationis performed by reacting the compound of formula (Va) with a base andwith a sulfinylation reagent. Non-limiting examples of sulfinylationreagent include methyl 2-pyridinesulfinate, methyl benzenesulfinate,methyl 4-methyl-benzenesufinate, and methyl 4-chloro-benzene sulfinate.The base used in the sulfinylation step can be selected from the groupcomprising potassium hydride, potassium terbutylate, sodium hydride,sodium terbutylate and a mixture thereof. Non-limiting examples ofsuitable experimental conditions for the sulfinylation are described inBarry M Trost et al in Journal of Organic Chemistry, 1993, 58, 1579-81;hereby incorporated by reference.

The next step (3) comprises the dehalogenation or desulfinylation of thecompound of formula (Vb) to produce compound of formula (V).

In an embodiment, step (2) is a halogenation, and step (3) comprises adehalogenation step which can be performed in the presence of a base.The base can be selected from the group comprising imidazole, collidine,2,6-lutidine, triethylamine, or 1,8-diazabicyclo[5.4.0]undec-7-ene(DBU). The dehalogenation reaction can be performed at a temperaturebetween 30° C. and 130° C. Preferably, the dehalogenation reaction isperformed in an aprotic solvent.

In another embodiment, step (2) is a sulfinylation, and step (3)comprises a desulfinylation which can be carried out with heatoptionally in the presence of cupric sulfate. The temperature of thedesulfinylation step can be between 80° C. and 130° C., preferablybetween 90° C. and 120° C., preferably between 100° C. and 115° C.

The next step (4) in the process comprises the reduction of the compoundof formula (Vb) with a reducing agent to produce compound of formula(II). Preferably, said reducing agent is a metal hydride compound. Forexample, the metal hydride compound can be selected from the groupcomprising LiAlH₄, NaBH₄, NaBH(OAc)₃, ZnBH₄, and NaBH₄/CeCl₃.Preferably, said reducing agent is NaBH₄/CeCl₃. For example saidreduction can be performed in a suitable solvent or a mixture thereof,such as in tetrahydrofuran, or a mixture of methanol andtetrahydrofuran. The reaction can be performed at low temperatures suchas below 15° C., for example below 10° C.

The present process for preparing compound (I) allows the preparation ofestra-1,3,5(10)-triene-3,15α,16α,17β-tetrol (sterol) as the majorproduct with little or no estra-1,3,5(10)-triene-3,15β,16β,17β-tetrolisomer being formed. As used herein little refers to obtaining more than90% of estetrol and less than 10% of the 15β,16β,17β-tetrol isomer,preferably less than 5% of the 15β,16β,17β-tetrol isomer, morepreferably less than 1% of the 15β,16β,17β-tetrol isomer.

The process according to the invention has the advantage that estetrol,can be obtained in a reduced number of steps compared to prior artprocesses, which is more convenient for an economical and industrialsynthesis.

The present invention also encompasses the use of estetrol directlyobtained by the process the invention for the manufacture of apharmaceutical composition, preferably for use in a method selected froma method of hormone replacement therapy, a method of treating vaginaldryness, a method of contraception, a method of enhancing libido, ofmethod of treating skin, a method of promoting wound healing, and amethod of treating or preventing a disorder selected from the groupconsisting of autoimmune diseases, breast tumors and colorectal tumors.

The invention is illustrated but not limited by the following examples.

EXAMPLES Example 1 Preparation of a Compound of Formula (I) Usingtert-butyl-dimethyl-silyl Group as Protecting Group for P¹ and P²According to an Embodiment of the Invention Step 1:estra-1,3,5(10),15-tetraene-3,17β-diol bis(dimethyl-tert-butylsilyl)ether

The starting material 3-t-butyldimethylsiloxy-estra-1,3,5(10)-15-tetraene-17β-ol can be prepared as described in Example 3 and 4.To a solution of 3-t-butyldimethylsiloxy-estra-1,3,5(10)-15-tetraene-17β-ol (10 g, 0.025 mole) in 100 ml ofdimethylformamide were added imidazole (4.4 g, 0.065 mole) anddimethyl-tert-butylsilyl-chloride (1.5 eq.) and allowed to stand at roomtemperature for 6 hours. The resulting solution was diluted with ethylacetate, washed with water and evaporated. The residue was crystallizedfrom methanol to afford (10 g) of estra-1,3,5(10),15-tetraene-3,17β-diolbis(dimethyl-tert-butylsilyl) ether.

NMR (CDCl₃): 0.08 (6H, s, 17-OSi(CH₃)₂, 0.18 (6H, 3-OSi(CH₃)₂, 0.81 (3H,s, 18-CH₃), 0.91 (9H, 17-OSi-t-Bu), 0.97 (9H, s, 3-OSi-t, Bu), 4.33 (1H,broad s, 17 aH), 5.60 (1H, m, 15-H, 5.95 (1H, broad, d, 16H), 6.45-6.75(2H, 2- and 4H), 7.12 (1H, d, J=8 Hz, 1H).

mp: 89-91° C.

Step 2: estra-1,3,5(10),15α,16α,17β-tetrol

To a stirred solution of estra-1,3,5(10),15-tetraene-3,17β-diolbis(dimethyl-tert-butylsilyl) ether (10 g, 0.02 mole) and formic acid(0.06 mole, 2.3 ml) in acetone (100 ml) at 0° C. was added gradually asolution of potassium permanganate (3.15 g, 0.02 mole) in water (20 ml)and acetone (100 ml). After completion of the reaction, the reaction wasquenched with a 10% aqueous solution of KHSO₃. Acetone was partiallyremoved and extracted with ethyl acetate, an washed with water. Ethylacetate was concentrated under reduced pressure and diluted withheptane. The precipitate was collected by filtration and dissolved inacetone (100 ml). To the solution 5N hydrochloric acid (20 ml) wasadded. After completion of the reaction the resulting solution wasdiluted with water. The solid was collected by filtration, washed withheptane and crystallized from a mixture of methanol and water to affordthe title compound.

Example 2 Preparation of a Compound of Formula (I) Usingtert-butyl-dimethyl-silyl Group as Protecting Group for P¹ and pivaloylfor P² According to an Embodiment of the Invention

The starting material 3-t-butyldimethylsiloxy-estra-1,3,5(10)-15-tetraene-17β-ol can be prepared as described in Example 3 and 4.To a solution of3-tert-butyldimethylsilyloxy-estra-1,3,5(10)-15-tetraene-17-ol (30 g,0.078 mole) in 300 ml of dichloromethane and 11 ml of triethylamine wereadded drop wise 10.36 g (0.086 mole) of pivaloyl chloride in 50 ml ofmethylene chloride at 0° C. At the end of the addition the solution wasstirred at room temperature for 1 hour. Water was added and the organiclayer was washed two time with 100 ml of water. Heptane was added andthe product was collected by filtration and used in the next stepwithout any other purification.

3-terbutyl-dimethylsilyloxy-estra-1,3,5(10)-15-tetraene-17β-pivaloatewas converted to its 15α,16α derivative following the proceduredescribed in example 1 step 1.

Then this3-terbutyl-dimethylsilyloxy-estra-1,3,5(10)-15α,16α-diol-triene-17β-pivaloate(10 g, 0.02 mole) and K₂CO₃ (2.76 g, 0.02 mole) was suspended inmethanol 200 ml and stirred for 4 hours at room temperature. Water 300ml was added and the mixture was neutralized with 0.1N HCl. The productwas collected by filtration and dried to afford 7.5 g (90% yield) of3-terbutyl-dimethylsilyloxy-estra-1,3,5(10)-triene-15α,16α,17β-triol.

Deprotection in acidic medium of the silyl protecting group wasperformed using the same conditions as described in example 1 step 2,and allowed this compound to be converted to estetrol in 90% yield

Example 3 Preparation of a Compound of Formula (II) Wherein P¹ ist-butyldimethylsilyl According to an Embodiment of the Invention Step 1:3,17-di-t-butyldimethylsiloxy-estra-1,3,5(10)-16-tetraene-17-ol

To a solution of estrone (50 g, 0.185 mole) and 2,6-lutidine (62 g, 0.58mole) in dichloromethane 400 ml was added drop wiset-butyl-dimethylsilyl-triflate (102.6 g, 0.39 mole). The solution wasstirred at room temperature for 6 hours. Water (300 ml) was added andthe organic layer was washed with a diluted solution of sodiumcarbonate. The dichloromethane solution was partially evaporated andethyl acetate was added. Diisopropyl ether was added to this solution.The mixture was stirred for 2 hours at 0° C. The precipitate wascollected by filtration and dried. 83 g of the title compound wereobtained (90% yield).

Step 2: 3-t-butyldimethylsiloxy-estra-1,3,5(10)-15-tetraene-17-one

To a solution of3,17-di-t-butyldimethylsiloxy-estra-1,3,5(10)-16-tetraene-17-ol 83 g(0.166 mole) in 400 ml of acetonitrile was added Pd(OAc)₂ 3.8 g (0.017mole) in an oxygen atmosphere. The mixture was stirred at 40° C. for 12hours then filtered through a pad of celite. A diluted solution ofsodium carbonate was added and the mixture was extracted with ethylacetate.

After concentration, diisopropyl ether was added and the mixture wasstirred at 0° C. for one hour. The product (54.7 g, 86% yield) wascollected by filtration and used in the next step without furtherpurification.

Step 3: 3-t-butyldimethylsiloxy-estra-1,3,5(10)-15-tetraene-17-ol

The collected material (54.7 g, 0.143 mole) was dissolved in THF 300 mland a solution of cerium chloride heptahydrate (53.3 g, 0.143 mole) inmethanol (300 ml) was added. The mixture was cooled to 0° C. sodiumborohydride (8.12 g, 0.213 mole, 1.5 eq) was added portion wise keepingthe temperature below 9° C. At this end of the addition the mixture wasstored for one hour then quenched by addition of a 2N HCl solution (100ml). The solution was partly evaporated in situ and water (4 L) wasadded. The precipitate was collected by filtration and dried. Aftercrystallization from a mixture of ethanol/diisopropyl ether the productwas collected by filtration and dried. It weighted 46.6 g (85% yield).

Example 4 Preparation of a Compound of Formula (II) Wherein P¹ ist-butyldimethylsilyl According to an Embodiment of the Invention Step 1:3-t-butyldimethylsiloxy-estra-1,3,5(10)-triene-17-one

To a solution of estrone (100 g, 0.37 mole) in 400 ml ofdichloromethane, imidazole (50.36 g, 0.74 mole) andt-butyl-dimethylsilyl chloride (61.3 g, 0.41 mole) were added Thesolution was stirred at room temperature for 24 hours. Then water (200ml) was added. The organic layer was partially evaporated anddiisopropyl ether added. The white solid formed was collected byfiltration and dried. It weighted 135.2 g, yield 95%, mp 172° C.

1H NMR (200 MHz): 7.12 (d, J=7.9 Hz, 1H), 6.61 (m, 2H), 2.84 (m, 3H),2.06-1.45 (m, 12H), 0.97 (s, 9H), 0.91 (s, 3H), 0.18 (s, 6H).

Step 2: 3-t-butyldimethylsiloxy-estra-1,3,5(10)-16-tetraene-17-acetate

3-t-butyldimethylsiloxy-estra-1,3,5(10)-triene-17-one 135 g (0.351 mole)were poured in 600 ml of isopropenyl acetate and 12 g ofpara-toluene-sulfonic acid. The mixture was refluxed. Acetone andisopropenyl acetate were continuously distilled off until the internaltemperature reached 98° C. Then the mixture was cooled to 0° C. andpotassium carbonate added. After one hour at 0° C. the mixture wasfiltered. The resulting solution was partially concentrated anddiisopropyl ether added. The precipitate was collected by filtration andcrystallized from a mixture of ethyl acetate and heptane. The productwas collected by filtration and dried. It weighted 119.5 g (yield 80%).

Step 3: 3-t-butyldimethylsiloxy-estra-1,3,5 (10)-15-tetraene-17-ol

To a solution of3-t-butyldimethylsiloxy-estra-1,3,5(10)-16-tetraene-17-acetate 119.5 g(0.280 mole) in acetonitrile (1500 ml) were added 27.2 g (0.085 mole oftributyltin methoxide, 11.2 g (0.05 mole) of palladium acetate and 64 ml(0.560 mole) of allyl methyl carbonate. The mixture was refluxed for 2hours then cooled to room temperature and filtered through a pad ofsilica gel. The mixture was diluted with water and extracted with ethylacetate. After concentration to one third of the initial volumediisopropyl ether was added and the solution cooled at 0° C. for onehour.

The product was collected by filtration. It weighted 91 g (85% yield)and was used in the next step without further purification.

Step 4: 3-t-butyldimethylsiloxy-estra-1,3,5(10)-15-tetraene-17-ol

The reduction step was performed as described in step 3 of example 2:the collected material was dissolved in THF and a solution of ceriumchloride heptahydrate (1 eq) in methanol was added. The mixture wascooled to 0° C. sodium borohydride (1.5 eq) was added portion wisekeeping the temperature below 9° C. At this end of the addition themixture was stored for one hour then quenched by addition of a 2N HClsolution. The solution was partly evaporated in situ and water wasadded. The precipitate was collected by filtration and dried. Aftercrystallization from a mixture of ethanol/diisopropyl ether the productwas collected by filtration and dried.

Example 5 Preparation of a Compound of Formula (II) Wherein P¹ istert-butyldimethylsilyl According to an Embodiment of the Invention Step1: 3-tert-butyldimethylsilyloxy-estra-1,3,5(10)-triene-17-one

To a solution of 3-hydroxy-estra-1,3,5(10)-triene-17-one (100 g, 0.370mole) in 500 ml of dichloromethane was addedtert-butyldimethylsilyl-chloride (58.3 g, 0.388 mole) and imidazole(26.4 g, 0.388 mole). The mixture was stirred for 24 hours at roomtemperature. Water (300 ml) was added and the organic layer was washedwith 200 ml of water. After concentration the product was crystallizedfrom a mixture of ethanol/diisopropyl ether, collected by filtration anddried. It weighted 145 g (95% yield).

Step 2: 3-tert-butyldimethylsilyloxy-estra-1,3,5(10)-15-tetraene-17-one

A solution of potassium terbutylate (50 g, 0.45 mole) in 800 ml oftetrahydrofuran was treated with3-tert-butyldimethylsilyloxy-estra-1,3,5(10)-triene-17-one (86.5 g,0.225 mole) under nitrogen and stirred for 1 hour, then methylbenzenesulfinate (70.2 g, 0.45 mole) and triethylamine were added. Afterstirring for 2 hours the solution was poured in 1000 ml of water and 70ml of hydrochloric acid keeping the temperature below 5° C. 1000 ml oftoluene was added, phases are separated and the solution was heated todistil off the solvent until the temperature reached 115° C. Reflux wasmaintained for 5 hours.

Toluene was washed with two time water, and then partially concentrated.Heptane was added. After one hour at 5° C. the solid was collected byfiltration and used in the reduction step without further purification.

Step 3: 3-tert-butyldimethylsilyloxy-estra-1,3,5(10)-15-tetraene-17-ol

The material collected in step 2 was dissolved in THF 300 ml and asolution of cerium chloride heptahydrate (123 g, 0.33 mole) in methanol(300 ml) was added. The mixture was cooled to 0° C. and sodiumborohybride (17.8 g, 0.47 mole, 1.5 q) was added portionwise keeping thetemperature below 9° C. At this end of the addition the mixture wasstirred for one hour then quenched by addition of a 2N HCl solution (100ml), extracted with ethyl acetate and washed with water. The organiclayer was partly evaporated then diisopropylether was added. Theprecipitate was collected by filtration and dried. After crystallizationform a mixture of ethanol/diisopropyl ether the title compound wasisolated in 90% yield as an off white solid.

Example 6 Preparation of a Compound of Formula (II) Wherein P¹ istert-butyldimethylsilyl According to an Embodiment of the Invention Step1: 3-tert-butyldimethylsilyloxy-estra-1,3,5(10)-triene-17-one

3-tert-butyldimethylsilyloxy-estra-1,3,5(10)-triene-17-one was preparedas described in step 1 of Example 5.

Step 2: 3-tert-butyldimethylsilyloxy-estra-1,3,5(10)-15-tetraene-17-one(via X═Br)

Copper(II) bromide (100 g, 0.45 mole) was added to a warm solution of3-tert-butyldimethylsilyloxy-estra-1,3,5(10)-triene-17-one (86.4 g,0.225 mole) in methanol (500 ml) and the mixture was heated under refluxfor 2 hours. The hot mixture was filtered and was poured in a mixture ofdichloromethane (1000 ml) and water (800 ml). The organic layer waswashed with water.

To this solution imidazole (18.3 g, 0.27 mole) was added and heatedunder reflux for 6 hours. After cooling water (500 ml) was added and theorganic layer was concentrated. The residue was crystallized from amixture of ethyl acetate and heptane.

Step 3: 3-tert-butyldimethylsilyloxy-estra-1,3,5(10)-15-tetraene-17-ol

The reduction step was performed as described in step 3 of example 1:The material collected in step 2 of example 2 was dissolved in THF and asolution of cerium chloride heptahydrate (about 1 eq) in methanol wasadded. The mixture was cooled to 0° C. and sodium borohybride (1.5 eq)was added portionwise keeping the temperature below 9° C. At this end ofthe addition the mixture was stirred for one hour then quenched byaddition of a 2N HCl solution, extracted with ethyl acetate and washedwith water. The organic layer was partly evaporated thendiisopropylether was added. The precipitate was collected by filtrationand dried. After crystallization form a mixture of ethanol/diisopropylether the title compound was isolated as an off white solid.

Example 7 Preparation of a Compound of Formula (II) Wherein P¹ istert-butyldimethylsilyl According to an Embodiment of the Invention Step1: 3-tert-butyldimethylsilyloxy-estra-1,3,5(10)-triene-17-one

3-tert-butyldimethylsilyloxy-estra-1,3,5(10)-triene-17-one was preparedas described in step 1 of Example 5.

Step 2: 3-tert-butyldimethylsilyloxy-estra-1,3,5(10)-15-tetraene-17-one(via X=pyridinesulfinic)

3-tert-butyldimethylsilyloxy-estra-1,3,5(10)-triene 17-one (8.64 g,0.0225 mole) was added to a suspension of potassium hydride (3 eq. 35%dispersion in oil) in tetrahydrofuran 100 ml. methyl 2-pyridinesulfinate(5.3 g, 0.034 mole, 1.5 eq) was added. After 30 min at room temperaturethe reaction was poured into a sulfate buffer. The aqueous phase wasneutralized by an aqueous solution of sodium carbonate then extractedwith toluene. The solution was heated to 110° C. for one hour. Aftercooling to room temperature the solution was washed with a dilutedsolution of sodium hydroxide then with water. The organic layer waspartly concentrated following by an addition of heptane. The3-tert-butyldimethylsilyloxy-estra-1,3,5(10)-15-tetraene-17-one wascollected by filtration.

Step 3: 3-tert-butyldimethylsilyloxy-estra-1,3,5(10)-15-tetraene-17-ol

The reduction step was performed as described in step 3 of example 1:The material collected in step 2 of example 3 was dissolved in THF and asolution of cerium chloride heptahydrate in methanol was added. Themixture was cooled to 0° C. and sodium borohybride (1.5 eq) was addedportionwise keeping the temperature below 9° C. At this end of theaddition the mixture was stirred for one hour then quenched by additionof a 2N HCl solution, extracted with ethyl acetate and washed withwater. The organic layer was partly evaporated then diisopropylether wasadded. The precipitate was collected by filtration and dried. Aftercrystallization form a mixture of ethanol/diisopropyl ether the titlecompound was isolated as an off white solid.

It is to be understood that although preferred embodiments and/ormaterials have been discussed for providing embodiments according to thepresent invention, various modifications or changes may be made withoutdeparting from the scope and spirit of this invention.

1. A process for the preparation of a compound of formula (I), or ahydrate or solvate thereof;

said process comprising the steps of a) reacting a compound of formula(II), with an acylating or a silylating agent to produce a compound offormula (III),

wherein P¹ is a protecting group selected from R¹CO—, or R²Si(R³)(R⁴)—,P² is a protecting group selected from (R⁶R⁵R⁷)C—CO—, or(R²)Si(R³)(R⁴)—, wherein R¹ is a group selected from C₁₋₆alkyl orC₃₋₆cycloalkyl, each group being optionally substituted by one or moresubstituents independently selected from fluoro or C₁₋₄alkyl; R², R³ andR⁴ are each independently a group selected from C₁₋₆alkyl or phenyl,each group being optionally substituted by one or more substituentsindependently selected from fluoro or C₁₋₄alkyl; R⁵ is a group selectedfrom C₁₋₆alkyl or phenyl, each group being optionally substituted by oneor more substituents independently selected from fluoro or C₁₋₄alkyl; R⁶and R⁷ are each independently hydrogen or a group selected fromC₁₋₆alkyl or phenyl, each group being optionally substituted by one ormore substituents independently selected from fluoro or C₁₋₄alkyl; b)reacting the compound of formula (III) in the presence of at least oneoxidizing agent selected from permanganate salt, osmium oxide, hydrogenperoxide, or iodine and silver acetate to produce compound of formula(IV); and

c) deprotecting the compound of formula (IV) to produce compound offormula (I).
 2. The process according to claim 1, wherein P¹ isR²Si(R³)(R⁴)—, and P² is (R²)Si(R³)(R⁴)—.
 3. The process according toclaim 1 or 2, wherein the silylating agent is selected fromC₁₋₆alkylsilylchloride, C₁₋₆alkylsilyltriflate, C₆arylsilyl chloride,C₆arylsilyltriflate, C₁₋₆alkylC6arylsilylchloride, orC₁₋₆alkylC6arylsilyltriflate, each group being optionally substituted byone or more substituents independently selected from fluoro orC₁₋₄alkyl.
 4. The process according to any one of claims 1 to 3, whereinthe acylating agent is selected from

wherein R⁵, R⁶, R⁷ have the same meaning as that defined in claim 1, R⁸is a group selected from C₁₋₆alkyl, or C₂₋₆alkenyl, each group beingoptionally substituted by one or more substituents independentlyselected from fluoro or C₁₋₄alkyl.
 5. The process according to any oneof claims 1 to 4, wherein in step (b) said oxidizing agent is potassiumpermanganate.
 6. The process according to claim 5, wherein step (b) isperformed in the presence of an acid.
 7. The process according to anyone of claims 1 to 6 wherein the compound of formula (II) is obtained bya process comprising the steps of: i) reacting a compound of formula(V), with an acylating or a silylating agent to produce a compound offormula (VI),

wherein P³ is a protecting group selected from R⁹CO—, orR¹⁰Si(R¹¹)(R¹²)—, wherein R⁹ is a group selected from C₁₋₆alkyl orC₃₋₆cycloalkyl, each group being optionally substituted by one or moresubstituents independently selected from fluoro or C₁₋₄alkyl; R¹⁰, R¹¹and R¹² are each independently a group selected from C₁₋₆alkyl orphenyl, each group being optionally substituted by one or moresubstituents independently selected from fluoro or C₁₋₄alkyl; ii)reacting the compound of formula (VI) in the presence of palladiumacetate or a derivative thereof, or iodine (V) species, to produce acompound of formula (VII); and

iii) reacting the compound of formula (VII) with a reducing agent toproduce the compound of formula (II).
 8. The process according to claim7, wherein P³ is R⁹CO—.
 9. The process according to claim 8, whereinstep (i) comprises the steps of (i1) protecting the hydroxyl of thecompound of formula (V) with a silylating agent to produce a compound offormula (Va), wherein P¹ has the same meaning as that defined in claim1; and

(i2) protecting the ketone of the compound of formula (Va) in thepresence of an acylating agent to produce compound of formula (VI). 10.The process according to any one of claims 1 to 6, wherein the compoundof formula (II) is obtained by a process comprising the steps of 1)reacting a compound of formula (V) with a silylating or an acylatingagent to produce a compound of formula (Va), wherein P¹ has the samemeaning as in claim 1;

2) halogenation or sulfinylation of the compound of formula (Va) toproduce a compound of formula (Vb);

wherein X is halo, or —O—SO—R²⁰, and R²⁰ is a group selected fromC₆₋₁₀aryl or heteroaryl, each group being optionally substituted by oneor more substituents independently selected from chloro or C₁₋₄alkyl; 3)dehalogenation or desulfinylation of the compound of formula (Vb) toproduce the compound of formula (V); and

4) reacting the compound of formula (VII) with a reducing agent toproduce the compound of formula (II).
 11. The process according to claim10, wherein step (2) is a sulfinylation and the sulfinylation isperformed by reacting the compound of formula (Va) with a base and witha sulfinylation reagent.
 12. The process according to claim 10, whereinstep (2) is a halogenation and the halogenation is performed by reactingthe compound of formula (Va) with a halogenating reagent.
 13. Theprocess according to any one of claims 7 and 10, wherein step (iii) andstep (4) are performed using a reducing agent selected from metalhydride compounds.
 14. Process according to any one of claims 7 to 13,wherein the silylating agent is selected from C₁₋₆alkylsilylchloride,C₁₋₆alkylsilyltriflate, C₆arylsilylchloride, C₆arylsilyltriflate,C₁₋₆alkylC₆arylsilylchloride, C₁₋₆alkylC₆arylsilyltriflate, each groupbeing optionally substituted by one or more substituents independentlyselected from fluoro or C₁₋₄alkyl.
 15. Process according to any one ofclaims 7 to 13, wherein the acylating agent is selected fromC₂₋₆alkenylC₁₋₆alkanoates, C₂₋₆alkenylC₃₋₆cycloalkanoate, acyl chloridesand anhydrides.